Ubiquitin-proteasome system is responsible for the degradation of nearly all regulatory proteins that are critical for maintaining basic cellular processes. The Lid is a sub-complex of the proteasome that is required for the degradation of ubiquitin-conjugated proteins. Despite this significance, it remains unclear why the Lid is indispensable during proteolysis. The only known function of the Lid is substrate deubiquitination, which is mediated by one of its nine subunits, Rpn11, and most Lid subunits have not been well characterized. Therefore, the central goal in this proposal is to understand Lid functions in protein breakdown by uncovering novel functions of the proteasome Lid. In specific Aim 1, a large-scale screen for mutations in each subunit of the Lid will be conducted using a novel genetic screen developed in the sponsoring lab that utilizes three reporters that are designed to be stabilized when mutations in the Lid subunits affect proteasome-dependent degradation. Since these reporters include nutritional markers and a drug resistance gene, Lid mutants can be rapidly selected for on plates lacking the appropriate.nutrient or drug. Candidate Lid mutants will be subsequently analyzed for various biochemical parameters of proteasome function including proteasome assembly and Rpn11 activity to elucidate how such mutations in a given Lid mutant affects protein degradation. Interestingly, Rpn 11-dependent Lid function requires the context of the entire proteasome. This suggests that the Lid-mediated control of the protein breakdown is coordinated with other regulations operated by the Base, specifically substrate unfolding, the nearest step that accompanies substrate deubiquitination. In specific Aim 2, a potential coupling of Lid activity to substrate unfolding will be examined. An unfolding assay based on hydrogen-deuterium exchange and mass spectrometry will be employed. This assay which was originally developed for prokaryotic ATP-dependent proteases can be easily modified for application to the eukaryotic proteasome. Results from this assay will help understand how the regulatory activities that occur in the Lid and the Base are coordinated during protein degradation. The ubiquitin-proteasome system has been implicated in various diseases including cancers. In fact, a general proteasome inhibitor, VELCADE has been used for the treatment of multiple myeloma. The functional investigation of the Lid will provide new insights into the regulations that occur during protein degradation and likely provide for the rationale design of a new generation of proteasome inhibitors that can target specific functions of the proteasome. [unreadable] [unreadable] [unreadable]